Project Summary/Abstract Polyomaviruses (PyVs) are small DNA tumor viruses that cause debilitating human disease, especially in immunocompromised individuals. To infect cells, these non-enveloped viruses must transport to the host nucleus where transcription and replication of the viral genome lead to lytic infection or cellular transformation. During entry, PyV sorts from the cell surface to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol. From here, the virus is disassembled in order to cross the narrow nuclear pore complex (NPC) and enter the nucleus. PyV transport from the cytosol to the nucleus is an important, yet enigmatic, step in infection. In mammalian cells, intracellular transport to the nucleus is facilitated largely by the cytoplasmic motor dynein, which moves cargo along microtubules towards the center of the cell. Using the prototypic PyV, simian virus 40 (SV40), which shares both structural and genetic organization with human PyVs as well as the same infectious life cycle, we recently reported that dynein motor activity is required for viral disassembly and nuclear arrival of the virus. The exact mechanisms by which dynein promotes this process are unknown. Processive dynein activity requires a three-protein complex composed of the dynein motor, dynactin activator, and an adaptor protein, which confers cargo specificity. Preliminary experiments reveal that in addition to dynein and dynactin, the bicaudal D2 (BICD2) cargo adaptor is also important for SV40 infection. The knockdown of BICD2 significantly impairs SV40 disassembly in the cytosol as well as its nuclear arrival. Moreover, BICD2 interacts directly with the virus and promotes its release at the NPC. In addition to activation by cargo adaptors, dynein activity can also be regulated by LIS1 with either NUDE or NDEL, co-factors that serve to anchor the motor to its microtubule track. Interestingly, we found that LIS1 and NDEL are also essential for SV40 disassembly and infection. This research proposal aims to define the role of dynein motor complex activators and regulators in the PyV entry pathway (Aim1), and whether the virus is directly capable of regulating this cellular process to complete its life cycle (Aim2). We hypothesize that SV40 recruits the dynein-dynactin-BICD2 (DDB) complex in the cytosol, which in turn transports the viral particle into the nucleus. Because the intact virus is too large to transport across the NPC, we further postulate that the coordinated action of dynein activators (BICD2) and regulators (LIS1/NDEL) produces a mechanical force that is strategically harnessed by the virus to generate a smaller core virus that can enter NPC. Upon completion of these studies, our findings will illuminate key steps in the PyV entry pathway and identify potential anti-viral targets for the prevention and treatment of PyV infection and disease.